Figure 1.

Diagram of the in vitro selection of endonucleases. (1) The DNA library (endonuclease gene in red) in the mix containing the PURE system is dispersed into aqueous droplets in a water-in-oil emulsion. Active endonuclease is expressed in vitro and cleaves the tail of its encoding gene at its recognition sequence (shown in red) in the droplets. (2) The reaction in the emulsion is quenched by heating and adding EDTA. The emulsion is broken by adding water-saturated ether. The DNA library is recovered from the aqueous phase. (3) Ligation is performed between the recovered DNA and an excess of a double-stranded adaptor with a compatible sticky end. (4) Adaptor-specific PCR is performed using the ligation mix. After PCR, the purified DNA either enters the next round of selection or proceeds to cloning and sequencing.